Chemokine Beta-15

ABSTRACT

The present invention concerns a member of the human chemokine CC protein family. In particular, isolated nucleic acid molecules are provided encoding the chemokine β-15 protein. Chemokine β-15 polypeptides are also provided. The invention further concerns diagnostic methods for detecting thymus disorders and therapeutic methods for modulating bone marrow cell proliferation and differentiation.

This application is a divisional of U.S. patent application Ser. No.10/263,766, filed Oct. 4, 2002, which is a divisional of U.S. patentapplication Ser. No. 09/272,162, filed Mar. 19, 1999, (now U.S. Pat. No.6,503,735), which is a continuation of U.S. patent application Ser. No.08/874,460, filed Jun. 16, 1997 (now U.S. Pat. No. 5,981,231), whichclaims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/019,837 filed on Jun. 17, 1996, each of which isherein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a human CC chemokine protein (i.e., acytokine having the first two of its four cysteine residues adjacent asindicated by “CC”) and to polynucleotides encoding this protein.

2. Background Information

The discovery of IL-8, in 1987, revealed the existence of a novel classof small cytokines, now called chemokines, that are widely studiedbecause of their ability to activate leukocytes and their potential roleas mediators of inflammations. A number of different human chemokineshave been identified after IL-8, by cloning or biochemical purificationand amino acid sequencing. All have four conserved cysteines that formcharacteristic disulfide bonds, a short amino-terminal and a longercarboxy-terminal sequence. Two subfamilies are distinguished by thearrangement of the first two cysteines, which are either separated byone amino acid (CXC chemokines) or are adjacent (CC chemokines.).Chemokine cDNAs typically encode proteins of 92-99 amino acids in lengththat are secreted after cleavage of a leader sequence of 20-25 aminoacids. Modeling on the basis of the NMR-derived structure of IL-8suggests that CXC and CC chemokines are folded in a similar manner.

The first human CC chemokine was identified by differentialhybridization cloning and was termed LD78 (Obaru, K. Fukuda, M., Maeda,S. and Shimada, K. (1986) J. Biochem. (Tokyo) 99, 885-894.) Several cDNAisoforms of a closely related human chemokine, Act-2, were laterdescribed (Miller, M. D. and Krangel, M. S. (1992) Crit. Rev. Immunol.12, 17-46), and two similar proteins, macrophage inflammatory protein 1α(MIP-1α) and MIP-1β, were purified form the culture medium oflipopolysaccharide (LPS)-stimulated mouse macrophages (Wolpe, S. D.,Davatelis, G. Sherry, B. et al. (1988) J. Exp. Med. 167, 570-581). Onthe basis of more than 70% amino acid identity, the murine and humanproteins are considered as homologs, and the terms human MIP-1α andMIP-1β are commonly used instead of LD78 and Act-2. The bestcharacterized CC chemokine is monocyte chemotactic protein 1 (MCP-1),which was purified and cloned from different sources (Miller, M. D. andKrangel, M. S. (1992) Crit. Rev. Immunol. 12, 17-46; Yoshimure, T.Robinson, E. A. Tanaka, S. Appella, E. and Leonardo, E. J. (1989) J.Immunol. 142, 1956-1962; Matsushima, K., Larsen, C. G., DuBois, G. C.and Oppenheim, J. J. (1989) J. Exp. Med. 169, 1485-1490). Other CCchemokines, 1-309 (Miller, M. D., Hata, S., De Waal Malafyt, R. andKrangel, M. S. (1989) J. Immunol. 143, 2907-2916), RANTES (Schall, T. J.Jongstra, J., Dyer, B. J. et al. (1988) J. Immunol. 141, 1018-1025) andHC14 (Chang, H. C., Hsu, F., Freeman, G. J., Griffin, J. D. andReinherz, E. L. (1989) Int. Immunol. 1, 388-397), were purified orcloned as products of activated T cells. HC14, termed MCP-2, was alsoisolated from osteosarcoma cell cultures (Van Damme, J. Proost, P.,Lenaerts, J-P. and Opdenakker, G. (1992) J. Exp. Med. 176, 59-65), alongwith a novel CC chemokine, MCP-3, which was subsequently cloned andexpressed (Minty, A. Chalon, P. Guillemot, J. C. et al. (1993) Eur.Cytokine Netw. 4, 99-110; Opdenakker, G. Froyen, G. Fiten, P., Proost,P. and Van Damme, J.(1993) Biochem. Biophys. Res. Commun. 1991,535-542). These CC chemokines share a sequence identify with MCP-1 ofbetween 29 and 71% (MCP-2 and MCP-3 have 62-71% identity with MCP-1).

MCP-1, the prototype of the CC chemokine sub-family, is chemotatic formonocytes but not for neutrophils (Yoshimure, T. Robinson, E. A. Tanaka,S. Appella, E. and Leonardo, E. J. (1989) J. Immunol. 142, 1956-1962;Matsushima, K., Larsen, C. G., DuBois, G. C. and Oppenheim, J. J. (1989)J. Exp. Med. 169, 1485-1490) and was initially considered to be acounterpart of IL-8. Indeed, monocytes respond to all CC chemokines, asjudged from stimulus-dependent [Ca²⁺]i changes (Miller, M. D. andKrangel, M. S. (1992) Crit. Rev. Immunol. 12, 17-46; Bioschoff, S. C.,Krieger, M. Brunner, T. et al. (1993) Eur. J. Immunol. 23, 761-767;McColl, S. R., Hachicha, M., Levasseur, S., Noete, K. and Schall, T. J.(1993) J. Immunol. 150, 4550-4560). MCP-1, MCP-2 and MCP-3 inducemonocyte infiltration on intradermal injection into rats and rabbits(Van Damme, J. Proost, P., Lenaerts, J-P. and Opdenakker, G. (1992) J.Exp. Med. 176, 59-65; Zacha, C. O. C., Anderson, A. O., Thompson, H. L.et al. (1990) J. Exp. Med. 171, 2177-2182), and MCP-1 also elicits inmonocytes a respiratory burst (Miller, M. D. and Krangel, M. S. (1992)Crit. Rev. Immunol. 12, 17-46) and the expression of β2 integrins(Jiang, Y., Beller, D. I., Frendl, G. and Graves, D. T. (1992) J.Immunol. 148, 2423-2428).

While the view that CXC chemokines act on neutrophils and CC chemokinesact on monocytes apparently remains valid, recent studies have revealedthat CC chemokines have a much wider range of biological activitiessince they can also activate some lymphocytes and, in particular,basophil and eosinophil leukocytes. Thus, there is a continuing need inthe art for isolating novel CC chemokines.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a human chemokine β-15 (CKβ-15)polypeptide having the amino acid sequence in FIGS. 1A-C (SEQ ID NO:2)or the amino acid sequence encoded by the cDNA clone deposited in abacterial host as ATCC Deposit Number 97519 on Apr. 25, 1996. Thenucleotide sequence determined by sequencing the deposited CKβ-15 cDNAclone, which is shown in FIGS. 1A-C (SEQ ID NO:1), contains an openreading frame encoding a polypeptide of about 149 amino acid residuesincluding an initiation codon at positions 1-3, a leader sequence ofabout 20 amino acid residues and a deduced molecular weight of about 16kDa. The 129 amino acid sequence of the predicted mature CKβ-15 proteinis shown in FIGS. 1A-C (amino acid residues from about 21 to about 149)and in SEQ ID NO:2 (amino acid residues from about 1 to about 129) andin SEQ ID NO:2 (amino acid residues from about 1-129).

Thus, one aspect of the invention provides isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the chemokine β-15 polypeptide having the complete amino acidsequence in SEQ ID NO:2; (b) a nucleotide sequence encoding thechemokine β-15 polypeptide having the complete amino acid sequence inSEQ ID NO:2 but lacking the N-terminal methionine residue; (c) anucleotide sequence encoding the mature chemokine β-15 polypeptidehaving the amino acid sequence at positions from about 1 to about 129 inSEQ ID NO:2; (d) a nucleotide sequence encoding, the chemokine β-15polypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC™ Deposit No. 97519; (e) a nucleotide sequenceencoding the mature chemokine β-15 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC™ Deposit No. 97519;and (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e) above. Preferably, the nucleicacid molecule will encode the mature polypeptide in SEQ ID NO:2 orencoded by the above-described deposited cDNA.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences. in (a), (b), (c),(d), (e), or (f) above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide having anucleotide sequence identical to a nucleotide sequence in (a), (b), (c),(d), (e), or (f), above. The polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues. An additional nucleic acid embodiment of the invention relatesto an isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of achemokine β-15 polypeptide having an amino acid sequence in (a), (b),(c), (d), or (e), above.

The present invention also relates to recombinant vectors which includethe isolated nucleic acid molecules of the present invention and to hostcells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofCKβ-15 polypeptides or peptides by recombinant techniques.

The invention further provides an isolated chemokine β-15 polypeptidehaving amino acid sequence selected from the group consisting of: (a) apolypeptide comprising amino acids from about −20 to about 129 in SEQ IDNO:2; (b) a polypeptide comprising amino acids from about −19 to about129 in SEQ ID NO:2; (c) a polypeptide comprising amino acids from about1 to about 129 in SEQ ID NO:2; (d) the amino acid sequence of thechemokine β-15 polypeptide having the complete amino acid sequenceincluding the leader encoded by the cDNA clone contained in ATCC™Deposit No. 97519; and (e) the amino acid sequence of the maturechemokine β-15 polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC™ Deposit No. 97519. The polypeptides of thepresent invention also include polypeptides having an amino acidsequence with at least 90% similarity, more preferably at least 95%similarity to those described in (a), (b), (c), (d), or (e) above, aswell as polypeptides having an amino acid sequence at least 80%identical, more preferably at least 90% identical, and still morepreferably 95%, 96%, 97%, 98% or 99% identical to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a chemokine β-15 polypeptide having an aminoacid sequence described in (a), (b), (c), (d) or (e), above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a chemokine β-15 polypeptide of the invention includeportions of such polypeptides with at least six or seven, preferably atleast nine, and more preferably at least about 30 amino acids to about50 amino acids, although epitope-bearing polypeptides of any length upto and including the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention. In anotherembodiment the invention provides an isolated antibody that bindsspecifically to a chemokine β-15 polypeptide having an amino acidsequence described in (a), (b), (c), (d) or (e) above.

The present inventors have discovered that CKβ-15 is expressed only intissue of the thymus. FIG. 3. For a number of thymus disorders,significantly higher or lower levels of CKβ-15 gene expression can bedetected in thymus tissue or bodily fluids (e.g., serum, plasma, urine,synovial fluid or spinal fluid) taken from an individual having such adisorder, relative to a “standard” CKβ-15 gene expression level, i.e.,the CKβ-15 expression level in thymus tissue or bodily fluids from anindividual not having the thymus disorder. Thus, the invention providesa diagnostic method useful during diagnosis of a thymus disorder, whichinvolves (a) assaying chemokine β-15 gene expression level in cells orbody fluid of that individual; (b) comparing that chemokine β-15 geneexpression level with a standard chemokine β-15 gene expression level,whereby an increase or decrease in the assayed chemokine β-15 geneexpression level compared to the standard expression level is indicativeof a thymus disorder. An additional aspect of the invention is relatedto a method for treatment of an individual in need of an increased levelof chemokine β-15 activity in the body comprising administering to suchan individual a composition comprising an isolated chemokine β-15polypeptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQID NO:2) sequences of the complete chemokine β-15 protein determined bysequencing of the DNA clone contained in ATCC™ Deposit No. 97519. Theprotein has a leader sequence of about 20 amino acid residues(underlined) and a deduced molecular weight of about 16 kDa. The aminoacid sequence of the predicted mature CKβ-15 protein is shown in FIGS.1A-C (last 129 amino acids) and in SEQ ID NO:2 (from amino acid residue1 to residue 129).

FIG. 2 shows the regions of similarity between the amino acid sequencesof the CKβ-15 protein and the mouse macrophage inflammatoryprotein-related protein 2 (MMRP-2) [SEQ ID NO:3].

FIG. 3 shows a Northern blot assay for expression of mRNA from theCKβ-15 gene in various human tissues. The panel labeled “HTSEX82” showshybridization to the CKβ-15 cDNA probe which that labeled “ACTIN” showshybridization of a cDNA encoding actin which serves as a positivecontrol indicating the presence of intact RNA in each sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the chemokine β-15 (CKβ-15) proteinhaving the amino acid sequence shown in FIGS. 1A-C (SEQ ID NO:2) whichwas determined by sequencing a cloned cDNA. CKβ-15 is a novel member ofthe β-chemokine subfamily (CC) whose genes are on human chromosome 17and on mouse chromosome 11 (Wilson, S. D., et al., J. Exp. Med.171:1301(1990) and Modi, W. S., et al., Hum. Genet. 84:185 (1990)). TheCKβ-15 protein of the present invention shares sequence homology withthe mouse macrophage inflammatory protein-related protein 2 (MMRP-2)(FIG. 2) (SEQ ID NO:3). The nucleotide sequence shown in FIGS. 1A-C (SEQID NO:1) was obtained by sequencing the HTSEX82 cDNA clone encoding aCKβ-15 polypeptide, which was deposited on Apr. 25, 1996 at the AmericanType Culture Collection, Patent Depository, 10801 University Boulevard,Manassas, Va. 20110-2209. The deposited clone is contained in thepBluescript™ SK(−) plasmid (Stratagene, LaJolla, Calif.).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain a some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U) where each thymidine deoxynucleotide (T) in the specifieddeoxynucleotide sequence in is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO:1 set forth using deoxyribonucleotide abbreviations isintended to indicate an RNA molecule having a sequence in which eachdeoxynucleotide A, G or C of SEQ ID NO:1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxynucleotide T hasbeen replaced by a ribonucleotide U.

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-C, a nucleic acid molecule of the present invention encodinga CKβ-15 polypeptide may be obtained using standard cloning andscreening procedures, such as those for cloning cDNAs using mRNA asstarting material. Illustrative of the invention, the nucleic acidmolecule described in FIGS. 1A-C (SEQ ID NO:1) was discovered in a cDNAlibrary derived from human thymus tissue. The determined nucleotidesequence of the CKβ-15 cDNA of FIGS. 1A-C contains an open reading frameencoding a protein of about 149 amino acid residues with an initiationcodon at positions 1-3 of the nucleotide sequence shown in FIGS. 1A-C(SEQ ID NO. 1), and a predicted leader sequence of about 20 amino acidresidues, and a deduced molecular weight of about 16 kDa. The amino acidsequence of the predicted mature CKβ-15 protein is shown in FIGS. 1A-Cfrom amino acid residue 21 to residue 149 and in SEQ ID NO:2 from aminoacid residue 1 to 129. The CKβ-15 protein shown in FIGS. 1A-C (SEQ IDNO:2) is about 34% identical and about 53% similar to MMRP2 (FIG. 2).

The present invention also provides the mature form(s) of the CKβ-15polypeptide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides a nucleotide sequence encodingthe mature CKβ-15 polypeptides having the amino acid sequence encoded bythe cDNA clone contained in the host identified as ATCC™ DepositNo.97519 and as shown in SEQ ID NO:2. By the mature CKβ-15 proteinhaving the amino acid sequence encoded by the cDNA clone contained inthe host identified as ATCC™ Deposit No. 97519 is meant the matureform(s) of the CKβ-15 polypeptide produced by expression in a mammaliancell (e.g., COS cells, as described below) of the complete open readingframe encoded by the human DNA sequence of the clone contained in thevector in the deposited host. As indicated below, the mature CKβ-15polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC™ Deposit No. 97519 may or may not differ from thepredicted “mature” CKβ-15 polypeptide shown in SEQ ID NO:2 (amino acidsfrom about 1 to about 129) depending on the accuracy of the predictedcleavage site based on computer analysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the methods of McGeoch, Virus Res. 3, 271-286 (1985) and vonHeinje, Nucleic Acids Res. 14, 4683-4690 (1986) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequence of the completeCKβ-15 polypeptides of the present invention were analyzed by a computerprogram (PSORT) (Nakai, K. and Kanehisa, M. Genomics 14, 897-911(1992)), which is an expert system for predicting the cellular locationof a protein based on the amino acid sequence. As part of thiscomputational prediction of localization, the methods of McGeoch and vonHeinje are incorporated. The analysis by the PSORT program predicted thecleavage site between amino acids −1 and 1 in SEQ ID NO:2. Thereafter,the complete amino acid sequences were further analyzed by visualinspection, applying a simple form of the (−1,−3) rule of von Heinje.von Heinje, supra. Thus, the leader sequence for the CKβ-15 polypeptideis predicted to consist of amino acid residues from about −20 to about−1 in SEQ ID NO:2, while the mature CKβ-15 polypeptide is predicted toconsist of residues from about 1 to about 129.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, as well as the variability ofcleavage sites for leaders in different known proteins, the actualCKβ-15 polypeptide encoded by the deposited cDNA comprises about 149amino acids, but may be anywhere in the range of 142-154 amino acids;and the actual leader sequence of this protein is about 20 amino acids,but may be anywhere in the range of about 15 to about 25 amino acids.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 1-3 of the nucleotide sequence shown in FIGS. 1A-C(SEQ ID NO:1); DNA molecules comprising the coding sequence for themature CKβ-15 protein shown in FIGS. 1A-C (amino acid residues fromabout 21 to about 149) and SEQ ID NO:2 (amino acid residues from about 1to about 129); and DNA molecules which comprise a sequence substantiallydifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode the CKβ-15 protein. Of course, thegenetic code is well known in the art. Thus, it would be routine for oneskilled in the art to generate the degenerate variants described above.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the CKβ-15 polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCC™Deposit No. 97519 on Apr. 25, 1996. Preferably, this nucleic acidmolecule will encode the mature polypeptide encoded by theabove-described deposited cDNA clone. In a further embodiment, nucleicacid molecules are provided encoding the full-length CKβ-15 polypeptidelacking the N-terminal methionine. The invention further provides anisolated nucleic acid molecule having the nucleotide sequence shown inFIGS. 1A-C (SEQ ID NO:1) or the nucleotide sequence of the CKβ-15 cDNAcontained in the above-described deposited clone, or nucleic acidmolecule having a sequence complementary to one of the above sequences.Such isolated molecules, particularly DNA molecules, are useful asprobes for gene mapping by in situ hybridization with chromosomes andfor detecting expression of the CKβ-15 gene in human tissue, forinstance, by Northern blot analysis. As described in detail below,detecting altered CKβ-15 gene expression in certain tissues or bodilyfluids is indicative of thymus disorders.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNA or the nucleotide sequence shown in SEQ ID NO:1 is intendedfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-500 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:1. Bya fragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in SEQ IDNO:1. Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the CKβ-15protein.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC™ Deposit 97519. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. By a polynucleotide whichhybridizes to a “portion” of a polynucleotide is intended apolynucleotide (either DNA or RNA) hybridizing to at least about 15nucleotides (nt), and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably about 30-70 ntof the reference polynucleotide. These are useful as diagnostic probesand primers as discussed above and in more detail below.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50-750 nt in length, or even to the entire length of thereference polynucleotide, also useful as probes according to the presentinvention, as are polynucleotides corresponding to most, if not all, ofthe nucleotide sequence of the deposited cDNA or the nucleotide sequenceas shown in FIGS. 1A-C (SEQ ID NO:1). By a portion of a polynucleotideof “at least 20 nt in length,” for example, is intended 20 or morecontiguous nucleotides from the nucleotide sequence of the referencepolynucleotide, (e.g., the deposited cDNA or the nucleotide sequence asshown in FIGS. 1A-C (SEQ ID NO:1)). As indicated, such portions areuseful diagnostically either as a probe according to conventional DNAhybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), ColdSpring Harbor Laboratory Press, the entire disclosure of which is herebyincorporated herein by reference.

Since a CKβ-15 cDNA clone has been deposited and its determinednucleotide sequence is provided in FIGS. 1A-C (SEQ ID NO:1), generatingpolynucleotides which hybridize to a portion of the CKβ-15 cDNA moleculewould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication of the CKβ-15 cDNA clonecould easily be used to generate DNA portions of various sizes which arepolynucleotides that hybridize to a portion of the CKβ-15 cDNA molecule.Alternatively, the hybridizing polynucleotides of the present inventioncould be generated synthetically according to known techniques. Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3, terminal poly(A) tract of the CKβ-15 cDNA shown in FIGS.1A-C (SEQ ID NO:1)), or to a complementary stretch of T (or U) resides,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule contain apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode the CKβ-15 protein polypeptide may include, but are not limitedto those encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 20 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro- protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing—including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.(1989) Proc. Natl. Acad. Sci., USA 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the CKβ-15polypeptide fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the CKβ-15 protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, ed. Non-naturally occurringvariants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the CKβ-15 protein or portions thereof.Also especially preferred in this regard are conservative substitutions.Most highly preferred are nucleic acid molecules encoding the matureCKβ-15 protein having the amino acid sequence shown in FIGS. 1A-C (SEQID NO:2) or the mature CKβ-15 amino acid sequence encoded by thedeposited cDNA clone.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the chemokine β-15polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b)a nucleotide sequence encoding the chemokine β-15 polypeptide having thecomplete amino acid sequence in SEQ ID NO:2 but lacking the N-terminalmethionine residue; (c) a nucleotide sequence encoding the maturechemokine β-15 polypeptide having the amino acid sequence at positionsfrom about 1 to about 129 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the chemokine β-15 polypeptide having the complete amino acidsequence encoded by the cDNA clone contained in ATCC™ Deposit No. 97519;(e) a nucleotide sequence encoding the mature chemokine β-15 polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC™ Deposit No. 97519; and (f) a nucleotide sequence complementary toany of the nucleotide sequences in (a), (b), (c), (d), or (e) above.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a chemokineβ-15 polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding thechemokine β-15 polypeptide. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIGS. 1A-C or to the nucleotides sequenceof the deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman(Advances in Applied Mathematics 2: 482-489, 1981) to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A-C (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA, irrespective of whether they encode a polypeptide havingCKβ-15 activity. This is because, even where a particular nucleic acidmolecule does not encode a polypeptide having CKβ-15 activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having CKβ-15 activityinclude, inter alia, (1) isolating the CKβ-15 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe CKβ-15 gene as described in Verma et al., Human Chromosomes: aManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting CKβ-15 mRNA expression in specifictissues (e.g., thymus tissue).

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A-C (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having CKβ-15protein activity. By “a polypeptide having CKβ-15 activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the CKβ-15 protein of the invention (either thefull-length protein or, preferably, the mature protein) as measured in aparticular biological assay. Like other CC cytokines, CKβ-15 exhibitsactivity on monocytes, lymphocytes and neutrophils. However, unlikeother known CC cytokines, CKβ-15 has been shown to be expressed only inthe thymus. Therefore, CKβ-15 is particularly active in modulatingactivities of cells in the thymus, particularly early thymocytes. Forexample, stimulation of early thymocyte proliferation by CKβ-15 isassayed in a standard proliferation assay (see, for instance, Spits etal. (1987) J. Immunol. 139:1142; Dalloul et al. (1989) Eur. J. Immunol.19:1985; Murphy et al., (1992) Ped. Res. 32:269; Ruggiero et al, (1996)J. Immunol. 156:3737). Briefly, the assay involves purification ofthymocytes from human thymus, plating them in media with or withoutCKβ-15, and determining the change with elapsed time in the rate ofproliferation or the number of cells compared to control cultures, byconventional means. Representative cell lines could also be employed insuch assays.

CKβ-15 also mediates the differentiation of intrathymic T cellprecursors into mature T-lymphocytes which are either α/β+ or γ/δ+ Tcell receptor lymphocytes (as defined in Barcena et al. (1990) J. Exp.Med. 172:439). This effect is mediated by modulating (either inducing orinhibiting) the apoptosis of specific subsets of thymocytes within thethymus or by directly inducing the differentiation of a specific subset.In addition CKβ-15 also directs the homing of the immature lymphocyteprecursor to the thymus for proper maturation. This activity isdemonstrated by in vitro chemotaxis assays using primary progenitors orrepresentative cell lines. CKβ-15 also mediates proper T-lymphocytematuration via the thymic epithelial cells, for example, by providing aco-stimulatory signal for proliferation or differentiation, as shown byvarious in vitro assays for human thymocyte proliferation ordifferentiation (Ruggiero et al. (1996) J. Immunol. 156:3737; Barcena etal. (1990) J. Exp. Med. 172:439; Singer et al. (1990) J. Immunol.144:2931).

The CKβ-15 protein of the present invention also modulates colonyformation of bone marrow progenitor cells, as does the macrophageinflammatory protein related protein-2 (MMRP-2). An in vitro colonyforming assay for measuring the extent of inhibition of myeloidprogenitor cells is described in Youn et al., The Journal of Immunology155:2661-2667 (1995). Briefly, the assay involves collecting human ormouse bone marrow cells and plating the same on agar, adding one or moregrowth factors and either (1) transfected host cell-supernatantcontaining CKβ-15 protein (or a candidate polypeptide) or (2)nontransfected host cell-supernatant control, and measuring the effecton colony formation by murine and human CFU-granulocyte-macrophages(CFU-GM), by human burst-forming unit-erythroid (BFU-E), or by human CFUgranulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM).

CKβ-15 protein modulates early thymocyte proliferation anddifferentiation in a dose-dependent manner in the above-describedassays. Thus, “a polypeptide having CKβ-15 protein activity” includespolypeptides that also exhibit any of the same thymocyte modulatingactivities in the above-described assays in a dose-dependent manner.Although the degree of dose-dependent activity need not be identical tothat of the CKβ-15 protein, preferably, “a polypeptide having CKβ-15protein activity” will exhibit substantially similar dose-dependence ina given activity as compared to the CKβ-15 protein (i.e., the candidatepolypeptide will exhibit greater activity or not more than about tenfoldless and, preferably, not more than about twofold less activity relativeto the reference CKβ-15 protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIGS. 1A-C (SEQ ID NO:1) willencode a polypeptide “having CKβ-15 protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving CKβ-15 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U., et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U., et al., supra, and the references cited therein.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of CKβ-15polypeptides or portions thereof by recombinant techniques.

Recombinant constructs may be introduced into host cells using wellknown techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include bacterial cells,such as E. coli, Streptomyces and Salmonella typhimurium cells; fungalcells, such as yeast cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanomacells; and plant cells. Appropriate culture media and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pA2, pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986).

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EPA 0 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EPA 0 232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when theFc portion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as an antigen forimmunizations. In drug discovery, for example, human proteins, such asthe hIL5-receptor, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. of Molec. Recognition 8:52-58 (1995) and K.Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

The CKβ-15 protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“BPLC”) is employed for purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

CKβ-15 Polypeptides and Peptides

The invention further provides an isolated CKβ-15 polypeptide having theamino acid sequence encoded by the deposited cDNA, or the amino acidsequence in FIGS. 1A-C (SEQ ID NO:2), or a peptide or polypeptidecomprising a portion of the above polypeptides. The terms “peptide” and“oligopeptide” are considered synonymous (as is commonly recognized) andeach term can be used interchangeably as the context requires toindicate a chain of at least two amino acids coupled by peptidyllinkages. The word “polypeptide” is used herein for chains containingmore than ten amino acid residues. All oligopeptide and polypeptideformulas or sequences herein are written from left to right and in thedirection from amino terminus to carboxy terminus.

It will be recognized in the art that some amino acid sequence of theCKβ-15 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of the CKβ-15polypeptide which show substantial CKβ-15 polypeptide activity or whichinclude regions of CKβ-15 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions (for example, substituting one hydrophilicresidue for another, but not strongly hydrophilic for stronglyhydrophobic as a rule). Small changes or such “neutral” amino acidsubstitutions will generally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr.

As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the CKβ-15 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the CKβ-15 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of the CKβ-15 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited cDNA minus the leader (i.e., themature protein); a polypeptide comprising amino acids from about −20 toabout 129 in SEQ ID NO:2; a polypeptide comprising amino acids fromabout −19 to about 129 in SEQ ID NO:2; a polypeptide comprising aminoacids from about 1 to about 129 in SEQ ID NO:2; as well as polypeptideswhich are at least 80% identical, more preferably at least 90% or 95%identical, still more preferably at least 96%, 97%, 98% or 99% identicalto the polypeptides described above and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2: 482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a chemokine β-15polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the chemokine β-15polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A-C (SEQ ID NO:2) or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

The polypeptide of the present invention are useful as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting CKβ-15 protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting CKβ-15 protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” CKβ-15 protein bindingproteins which are also candidate agonist and antagonist according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen, H. M., Meloen, R. H. and Barteling, S. J. (1984)Use of peptide synthesis to probe viral antigens for epitopes to aresolution of a single amino acid. Proc. Natl. Acad. Sci. USA81:3998-4002.

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Peptides that are extremely hydrophobic and those ofsix or fewer residues generally are ineffective at inducing antibodiesthat bind to the mimicked protein; longer, soluble peptides, especiallythose containing proline residues, usually are effective. Sutcliffe etal., supra, at 661. For instance, 18 of 20 peptides designed accordingto these guidelines, containing 8-39 residues covering 75% of thesequence of the influenza virus hemagglutinin HA1 polypeptide chain,induced antibodies that reacted with the HA1 protein or intact virus;and 12/12 peptides from the MuLV polymerase and 18/18 from the rabiesglycoprotein induced antibodies that precipitated the respectiveproteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes posttranslationprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance,Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R.,Connolly, M. L. and Lerner, R. A. (1984) The structure of an antigenicdeterminant in a protein. Cell 37:767-778 at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500-1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M.,Yabrov, R., Bittle, J., Hogel, J. and Baltimore, D., Proc. Natl. Acad.Sci. USA 82:910-914; and Bittle, F. J., Fry, C. M., Rowlands, D. J.,Brown, F., Bittle, J. L., Houghten, R. A. and Lerner, R. A. (1985) J.Gen. Virol. 66:2347-2354. Generally, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingof the peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., 1984, supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

Thymus-Related Disorder Diagnosis

The present inventors have discovered that CKβ-15 is expressed only inthymus tissue. For a number of thymus-related disorders, substantiallyaltered (increased or decreased) levels of CKβ-15 gene expression can bedetected in thymus tissue or other cells or bodily fluids (e.g., sera,plasma, urine, synovial fluid or spinal fluid) taken from an individualhaving such a disorder, relative to a “standard” CKβ-15 gene expressionlevel, that is, the CKβ-15 expression level in thymus tissue or bodilyfluids from an individual not having the thymus disorder. Thus, theinvention provides a diagnostic method useful during diagnosis of athymus disorder, which involves measuring the expression level of thegene encoding the CKβ-15 protein in thymus tissue or other cells or bodyfluid from an individual and comparing the measured gene expressionlevel with a standard CKβ-15 gene expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a thymus disorder.

By individual is intended mammalian individuals, preferably humans. By“measuring the expression level of the gene encoding the CKβ-15 protein”is intended qualitatively or quantitatively measuring or estimating thelevel of the CKβ-15 protein or the level of the mRNA encoding the CKβ-15protein in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level or mRNA level) orrelatively (e.g., by comparing to the CKβ-15 protein level or mRNA levelin a second biological sample). Preferably, the CKβ-15 protein level ormRNA level in the first biological sample is measured or estimated andcompared to a standard CKβ-15 protein level or mRNA level, the standardbeing taken from a second biological sample obtained from an individualnot having the disorder or being determined by averaging levels from apopulation of individuals not having a disorder of the thymus. As willbe appreciated in the art, once a standard CKβ-15 protein level or mRNAlevel is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains CKβ-15 protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain secreted mature CKβ-15 protein, thymustissue, and other tissue sources found to express CKβ-15 or a CKβ-15receptor. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

The present invention is useful for diagnosis or treatment of variousthymus-related disorders in mammals, preferably humans. Such disordersinclude the following tumors and cancers, hypoactivity, hyperactivity,atrophy, enlargement of the thymus, and the like. Other disordersinclude disregulation of T-lymphocyte selection or activity and wouldinclude but not be limited to disorders involving autoimmunity,arthritis, leukemias, lymphomas, immunosuppression, sepsis, wouldhealing, acute and chronic inflammation, cell mediated immunity, humorimmunity, TH1/TH2 imbalance, and the like.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the CKβ-15 protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al.,Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. CKβ-15 protein cDNAlabeled according to any appropriate method (such as the ³²P-multiprimedDNA labeling system (Amersham)) is used as probe. After hybridizationovernight, the filter is washed and exposed to x-ray film. cDNA for useas probe according to the present invention is described in the sectionsabove and will preferably at least 15 bp in length.

S1 mapping can be performed as described in Fujita et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe CKβ-15 protein). Northern blot analysis can be performed asdescribed above.

Preferably, levels of mRNA encoding the CKβ-15 protein are assayed usingthe RT-PCR method described in Makino et al., Technique 2:295-301(1990). By this method, the radioactivities of the “amplicons” in thepolyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the CKβ-15protein) is quantified using an imaging analyzer. RT and PCR reactioningredients and conditions, reagent and gel concentrations, and labelingmethods are well known in the art. Variations on the RT-PCR method willbe apparent to the skilled artisan.

Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

Assaying CKβ-15 protein levels in a biological sample can occur usingany art-known method. Preferred for assaying CKβ-15 protein levels in abiological sample are antibody-based techniques. For example, CKβ-15protein expression in tissues can be studied with classicalimmunohistological methods. In these, the specific recognition isprovided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of CKβ-15 protein for Western-blot or dot/slot assay(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985)); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). In this technique, whichis based on the use of cationic solid phases, quantitation of CKβ-15protein can be accomplished using isolated CKβ-15 protein as a standard.This technique can also be applied to body fluids. With these samples, amolar concentration of CKβ-15 protein will aid to set standard values ofCKβ-15 protein content for different body fluids, like serum, plasma,urine, synovial fluid, spinal fluid, etc. The normal appearance ofCKβ-15 protein amounts can then be set using values from healthyindividuals, which can be compared to those obtained from a testsubject.

Other antibody-based methods useful for detecting CKβ-15 protein levelsinclude immunoassays, such as the enzyme linked immunosorbent assay(ELISA) and the radioimmunoassay (RIA). For example, CKβ-15protein-specific monoclonal antibodies can be used both as animmunoadsorbent and as an enzyme-labeled probe to detect and quantifythe CKβ-15 protein. The amount of CKβ-15 protein present in the samplecan be calculated by reference to the amount present in a standardpreparation using a linear regression computer algorithm. Such an ELISAfor detecting a tumor antigen is described in Iacobelli et al., BreastCancer Research and Treatment 11:19-30 (1988). In another ELISA assay,two distinct specific monoclonal antibodies can be used to detect CKβ-15protein in a body fluid. In this assay, one of the antibodies is used asthe immunoadsorbent and the other as the enzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting CKβ-15protein with immobilized antibody and, without washing, contacting themixture with the labeled antibody. The “two-step” assay involves washingbefore contacting the mixture with the labeled antibody. Otherconventional methods may also be employed as suitable. It is usuallydesirable to immobilize one component of the assay system on a support,thereby allowing other components of the system to be brought intocontact with the component and readily removed from the sample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

In addition to assaying CKβ-15 protein levels in a biological sampleobtained from an individual, CKβ-15 protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of CKβ-15protein include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A CKβ-15 protein-specific antibody or antibody portion which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a thymus disorder.It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moietiesneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of ^(99m)Tc. The labeledantibody or antibody portion will then preferentially accumulate at thelocation of cells which contain CKβ-15 protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Portions” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, eds., S. W. Burchiel andB. A. Rhodes, Masson Publishing Inc. (1982)).

CKβ-15-protein specific antibodies for use in the present invention canbe raised against the intact CKβ-15 protein or an antigenic polypeptideportion thereof, which may presented together with a carrier protein,such as an albumin, to an animal system (such as rabbit or mouse) or, ifit is long enough (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody portions (suchas, for example, Fab and F(ab′)₂ portions) which are capable ofspecifically binding to CKβ-15 protein. Fab and F(ab′)₂ portions lackthe Fc portion of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, theseportions are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the CKβ-15 protein oran antigenic portion thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of CKβ-15 protein is prepared andpurified as described above to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or CKβ-15 protein binding portions thereof).Such monoclonal antibodies can be prepared using hybridoma technology(Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol.6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981)). In general, such procedures involve immunizing ananimal (preferably a mouse) with a CKβ-15 protein antigen or, morepreferably, with a CKβ-15 protein-expressing cell. Suitable cells can berecognized by their capacity to bind anti-CKβ-15 protein antibody. Suchcells may be cultured in any suitable tissue culture medium; however, itis preferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 μg/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP₂O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the CKβ-15 antigen.

Alternatively, additional antibodies capable of binding to the CKβ-15protein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, CKβ-15 protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theCKβ-15 protein-specific antibody can be blocked by the CKβ-15 proteinantigen. Such antibodies comprise anti-idiotypic antibodies to theCKβ-15 protein-specific antibody and can be used to immunize an animalto induce formation of further CKβ-15 protein-specific antibodies.

It will be appreciated that Fab and F(ab′)₂ and other portions of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such portions are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab portions) orpepsin (to produce F(ab′)₂ portions). Alternatively, CKβ-15protein-binding portions can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

Where in vivo imaging is used to detect enhanced levels of CKβ-15protein for diagnosis in humans, it may be preferable to use “humanized”chimeric monoclonal antibodies. Such antibodies can be produced usinggenetic constructs derived from hybridoma cells producing the monoclonalantibodies described above. Methods for producing chimeric antibodiesare known in the art. See, for review, Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat.No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

Further suitable labels for the CKβ-15 protein-specific antibodies ofthe present invention are provided below. Examples of suitable enzymelabels include malate dehydrogenase, staphylococcal nuclease,delta-S-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerolphosphate dehydrogenase, triose phosphate isomerase, peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging (Perkins et al., Eur. J. Nucl. Med. 10:296-301 (1985);Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)). For example, illincoupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumorous tissues, particularly the liver,and therefore enhances specificity of tumor localization (Esteban etal., J. Nucl. Med. 28:861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷ Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescarnine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and Fe.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al. (Clin. Chim. Acta 70:1-31 (1976)), andSchurs et al. (Clin. Chim. Acta 81:1-40 (1977)). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a CKβ-15 protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose. Typically, in accordance with routine procedures forchromosome mapping, some trial and error may be necessary to identify agenomic probe that gives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified portion.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of portions from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,MENDELIAN INHERITANCE IN MAN, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Treatment of Thymus-Related Disorders

As noted above, unlike other known CC cytokines, CKβ-15 has been shownto be expressed only in the thymus. Therefore, CKβ-15 is particularlyactive in modulating activities of monocytes in the thymus, particularlythose of early thymocytes, such as the activities described above inrelation to the description of a “polypeptide having CKβ-15 activity.”Given the thymocyte activities modulated by CKβ-15, it is readilyapparent that a substantially altered (increased or decreased) level ofexpression of CKβ-15 in an individual compared to the standard or“normal” level produces pathological conditions such as those describedabove in relation to diagnosis of thymus-related disorders. It will alsobe appreciated by one of ordinary skill that, since the CKβ-15 proteinof the invention is translated with a leader peptide suitable forsecretion of the mature protein from the cells which express CKβ-15,when CKβ-15 protein (particularly the mature form) is added from anexogenous source to cells, tissues or the body of an individual, theprotein will exert its modulating activities on any of its target cellsof that individual. Therefore, it will be appreciated that conditionscaused by a decrease in the standard or normal level of CKβ-15 activityin an individual, particularly disorders of the thymus, can be treatedbe administration of CKβ-15 protein. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofCKβ-15 activity comprising administering to such an individual apharmaceutical composition comprising an amount of an isolated CKβ-15polypeptide of the invention, particularly a mature form of the CKβ-15protein of the invention, effective to increase the CKβ-15 activitylevel in such an individual.

In addition, since the CKβ-15 protein suppresses myeloid cell growthwhen administered to an individual, the invention provides methods forsuppressing myeloid cell proliferation in an individual, which involveadministering a myelosuppressive amount of CKβ-15 either alone ortogether with one or more chemokines selected from the group consistingof Macrophage Inflammatory Protein-1α (MIP-1α), Macrophage InflammatoryProtein-2α (MIP-2α), Platelet Factor 4 (PF4), Interleukin-8 (IL-8),Macrophage Chemotactic and Activating Factor (MCAF), and MacrophageInflammatory Protein-Related Protein-2 (MRP-2). The myelosuppressivecompositions of the present invention thus provide myeloprotectiveeffects and are useful in conjunction with therapies that have anadverse affect on myeloid cells. This is because the myelosuppressivecompositions of the present invention place myeloid cells in aslow-cycling state thereby providing protection against cell damagecaused by, for example, radiation therapy or chemotherapy usingcell-cycle active drugs, such as cytosine arabinoside and hydroxyurea.

The myelosuppressive pharmaceutical compositions of the presentinvention are also useful in the treatment of leukemia, which causes ahyperproliferative myeloid cell state. Thus, the invention furtherprovides methods for treating leukemia, which involve administering to aleukemia patient a myelosuppressive amount of CKβ-15 either alone ortogether with one or more chemokines selected from the group consistingof MIP-1α, MIP-2α, PF4, IL-8, MCAF, and MRP-2.

By “suppressing myeloid cell proliferation” is intended decreasing thecell proliferation of myeloid cells and/or increasing the percentage ofmyeloid cells in the slow-cycling phase. As above, by “individual” isintended mammalian individuals, preferably humans. Preincubation of themyelosuppressive compositions of the present invention with acetonitrile(ACN) significantly enhances the specific activity of these chemokinesfor suppression of myeloid progenitor cells. Thus, preferably, prior toadministration, the myelosuppresive compositions of the presentinvention are pretreated with ACN as described in Broxmeyer H. E., etal., Ann-Hematol. 71(5):235-46(1995) and PCT Publication WO 94/13321,the entire disclosures of which are hereby incorporated herein byreference.

The myelosuppressive compositions of the present invention may be usedin combination with a variety of chemotherapeutic agents includingalkylating agents such as nitrogen mustards, ethylenimines,methylmelamines, alkyl sulfonates, nitrosuoureas, and triazenes;antimetabolites such as folic acid analogs, pyrimidine analogs, inparticular fluorouracil and cytosine arabinoside, and purine analogs;natural products such as vinca alkaloids, epipodophyllotoxins,antibiotics, enzymes and biological response modifiers; andmiscellaneous products such as platinum coordination complexes,anthracenedione, substituted urea such as hydroxyurea, methyl hydrazinederivatives, and adrenocorticoid suppressant.

Chemotherapeutic agents can be administered at known concentrationsaccording to known techniques. The myelosuppressive compositions of thepresent invention can be co-administered with a chemotherapeutic agent,or administered separately, either before or after chemotherapeuticadministration.

Certain chemokines, such as MIP-1β, MIP-2β and GRO-α, inhibit (at leastpartially block) the myeloid suppressive affects of the myelosuppresivecompositions of the present invention. Thus, in a further embodiment,the invention provides methods for inhibiting myelosuppression, whichinvolves administering an effective amount of a myelosuppressiveinhibitor selected from the group consisting of MIP-1β, MIP-2β and GRO-αto a mammal previously exposed to the myelosuppresive agent CKβ-15either alone or together with one or more of MIP-1α, MIP-2α, PF4, IL-8,MCAF, and MRP-2.

One of ordinary skill will appreciate that effective amounts of theCKβ-15 polypeptides for treating an individual in need of an increasedlevel of CKβ-15 activity (including amounts of CKβ-15 polypeptideseffective for myelosuppression with or without myelosuppressive agentsor myelosuppressive inhibitors) can be determined empirically for eachcondition where administration of CKβ-15 is indicated. The polypeptidehaving CKβ-15 activity my be administered in pharmaceutical compositionsin combination with one or more pharmaceutically acceptable excipients.It will be understood that, when administered to a human patient, thetotal daily usage of the pharmaceutical compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the type and degree of the response to be achieved; thespecific composition an other agent, if any, employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thecomposition; the duration of the treatment; drugs, (such as achemotherapeutic agent) used in combination or coincidental with thespecific composition; and like factors well known in the medical arts.

For example, satisfactory results are obtained by oral administration ofa polypeptide having CKβ-15 activity in dosages on the order of from0.05 to 10 mg/kg/day, preferably 0.1 to 7.5 mg/kg/day, more preferably0.1 to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 timesper day. On administration parenterally, for example by i.v. drip orinfusion, dosages on the order of from 0.01 to 5 mg/kg/day, preferably0.05 to 1.0 mg/kg/day and more preferably 0. 1 to 1.0 mg/kg/day can beused. Suitable daily dosages for patients are thus on the order of from2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.

Dosaging may also be arranged in a patient specific manner to provide apredetermined concentration of an CKβ-15 activity in the blood, asdetermined by an RIA technique, for instance. Thus patient dosaging maybe adjusted to achieve regular on-going trough blood levels, as measuredby RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500ng/ml.

Pharmaceutical compositions of the invention may be administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

Pharmaceutical compositions of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylceuulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

The compositions of the present invention may also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents, anddispersing agents. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents such as sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the pharmaceuticalcomposition, it is desirable to slow the absorption of the drug fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsuled matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompounds are mixed with at least one item pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcerulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f)absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin andbentonite clay, and I) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isoptopyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

The active polypeptide can also be administered in the form ofliposomes. As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to theagent or inhibitor, stabilizers, preservatives, excipients, and thelike. The preferred lipids are the phospholipids and the phosphatidylcholates (lecithins), both natural and synthetic. Methods to formliposomes are known in the art. See, for example, Prescott, Ed., Methodsin Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p.33 et seq.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of CKβ-15 in E. coli

The DNA sequence encoding the mature CKβ-15 protein in the depositedcDNA clone was amplified using PCR oligonucleotide primers specific tothe amino terminal sequences of the CKβ-15 protein and to vectorsequences 3′ to the gene. Additional nucleotides containing restrictionsites to facilitate cloning were added to the 5′ and 3′ sequencesrespectively.

The 5′ oligonucleotide primer had the sequence 5′ GCC GTC GAC GTC CACACC CAA GGT GTC 3′ (SEQ ID NO:4) containing the underlined SalIrestriction site, which encodes 18 nucleotides of the CKβ-15 proteincoding sequence in FIGS. 1A-C (SEQ ID NO:1) beginning immediately afterthe signal peptide.

The 3′ primer had the sequence 5′ GCC TCT AGA GGA GCC CAG AAA TGA GCCGGC 3′ [SEQ ID NO:5] containing the underlined XbaI restriction sitefollowed by 21 nucleotides complementary to the last 21 nucleotidesimmediately after the CKβ-15 protein coding sequence in FIGS. 1A-C.

The restriction sites were convenient to restriction enzyme sites in thebacterial expression vector pD10 (pQE9), which were used for bacterialexpression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). [pD10]pQE9 encodes ampicillin antibioticresistance (“Amp^(r)”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites.

The amplified CKβ-15 protein DNA and the vector pQE9 both were digestedwith SalI and XbaI and the digested DNAs were then ligated together.Insertion of the CKβ-15 protein DNA into the restricted pQE9 vectorplaced the CKβ-15 protein coding region downstream of and operablylinked to the vector's IPTG-inducible promoter and in-frame with aninitiating AUG appropriately positioned for translation of CKβ-15protein.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”),was used in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressingCKβ-15 protein, is available commercially from Qiagen.

Transformants were identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA was isolated fromresistant colonies and the identity of the cloned DNA was confirmed byrestriction analysis.

Clones containing the desired constructs were grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2× PBS at a concentration of 95 μ/ml.

Analysis of the preparation by standard methods of polyacrylamide gelelectrophoresis reveals that the preparation contains about 95% monomerCKβ-15 protein having the expected molecular weight of approximately16.7 kDa.

Example 2 Cloning and Expression of CKβ-15 Protein in a BaculovirusExpression System

The cDNA sequence encoding the full length CKβ-15 protein in thedeposited clone is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ GCC TCT AGA GCC ATC ATG AAC CTG TGGCTC CTG GCC 3′ (SEQ ID NO:6) containing the underlined XbaI restrictionenzyme site followed by 21 bases of the sequence of CKβ-15 protein inFIGS. 1A-C. Inserted into an expression vector, as described below, the5′ end of the amplified fragment encoding CKβ-15 provides an efficientsignal peptide. An efficient signal for initiation of translation ineukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950(1987) is appropriately located in the vector portion of the construct.

The 3′ primer has the sequence 5′ GCC TCT AGA GGA GCC CAG AAA TGA CCCGGC 3′ (SEQ ID NO:7) containing the underlined XbaI restriction sitefollowed by nucleotides complementary to the last 21 nucleotides of theCK[-15 coding sequence set out in FIGS. 1A-C.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with XbaI and again is purifiedon a 1% agaroae gel. This fragment is designated herein F2.

The vector pA2 is used to express the CKβ-15 protein in the baculovirusexpression system, using standard methods, as described in Summers etal, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTUREPROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555(1987). This expression vector contains the strong polyhedrin promoterof the Autographa californica nuclear polyhedrosis virus (AcMNPV)followed by convenient restriction sites such as BamHI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170:31-39, among others.

The plasmid is digested with the restriction enzyme XbaI and then isdephosphorylated using calf intestinal phosphatase, using routineprocedures known in the art. The DNA is then isolated from a 1% agarosegel using a commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated herein “V2”.

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli BB 101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human CKβ-15 gene by digesting DNA from individualcolonies using XbaI and then analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing. This plasmid is designated herein pBacCKβ-15.

5 μg of the plasmid pBacCKβ-15 is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBacCKβ-15 are mixed in a sterile well of a microtiter plate containing50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC™ CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is rocked back andforth to mix the newly added solution. The plate is then incubated for 5hours at 27° C. After 5 hours the transfection solution is removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. The plate is put back into an incubator andcultivation is continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted hESSB I, II and III is identified by DNAanalysis including restriction mapping and sequencing. This isdesignated herein as V-CKβ-15.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-CKβ-15 at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3 Expression in Mammalian Cells (COS)

The expression plasmid, pCKβ-15 HA, is made by cloning a cDNA encodingCKβ-15 into the expression vector pcDNAI/Amp (which can be obtained fromInvitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

A DNA fragment encoding the entire CKβ-15 precursor and an HA tag fusedin frame to its 3′ end is cloned into the polylinker region of thevector so that recombinant protein expression is directed by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is as follows.

The CKβ-15 cDNA of the deposited clone is amplified using primers thatcontain convenient restriction sites, much as described above regardingthe construction of expression vectors for expression of CKβ-15 in E.coli. To facilitate detection, purification and characterization of theexpressed CKβ-15, one of the primers contains a hemagglutinin tag (“HAtag”) as described above.

Suitable primers include that following, which are used in this example.The 5′ primer, containing the underlined HindIII site, an AUG startcodon and 6 codons of the 5′ coding region has the following sequence:5′ GCG AAG CTT ATG AAC CTG TGG CTC CTG GCC 3′ [SEQ ID NO:8].

The 3′ primer, containing the underlined XhoI site, a stop codon, 9codons thereafter forming the hemagglutinin HA tag, and 22 bp of 3′coding sequence (at the 3′ end) has the following sequence: 5′ GCG CTCGAG TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CAG TCC TGA ATT AGC TGA TATC 3′ [SEQ ID NO:9].

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith HindIII and XhoI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the CBβ-15-encoding fragment.

For expression of recombinant CKβ-15, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of CKβ-15 by thevector.

Expression of the CKβ-15 HA fusion protein is detected by radiolabellingand immunoprecipitation, using methods described in, for example Harlowet al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4 Tissue Distribution of CKβ-15 Protein Expression

Northern blot analysis was carried out to examine the levels ofexpression of CKβ-15 protein in human tissues, using methods describedby, among others, Sambrook et al, cited above. PolyA⁺ was purchased fromClontech (1020 East Meadow Circle, Palo Alto, Calif. 94303).

About 1 μg of PolyA⁺ RNA was size resolved by electrophoresis through a1% agarose gel under strongly denaturing conditions. RNA was blottedfrom the gel onto a nylon filter, and the filter then was prepared forhybridization to a detectably labeled polynucleotide probe.

As a probe to detect mRNA that encodes CKβ-15 protein, the antisensestrand of the coding region of the cDNA insert in the deposited clonewas labeled to a high specific activity. The cDNA was labeled by primerextension, using the Prime-It kit, available from Stratagene. Thereaction was carried out using 50 ng of the cDNA, following the standardreaction protocol as recommended by the supplier. The labeledpolynucleotide was purified away from other labeled reaction componentsby column chromatography using a Select-G-50 column, obtained from5-Prime -3-Prime, Inc. of 5603 Arapahoe Road, Boulder, Colo. 80303.

The labeled probe was hybridized to the filter, at a concentration of1,000,000 cpm/ml, as described in Kreider et al., Molecular and CellularBiology, September 1990, pp. 4846-4853. Thereafter the probe solutionwas drained and the filter was washed twice at room temperature andtwice at 65° C. with 0.1×SSC, 0.1% SDS. The filter then was then driedand exposed to film at −70° C. overnight with an intensifying screen.The results of a typical Nothern blot using the CKβ-15 cDNA probe areshown in FIG. 3.

Example 5 Gene Therapeutic Expression of Human CKβ-15 Protein

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted—the chunks of tissue remain fixed to the bottom of theflask—and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes forcloning a portion to be expressed. The digested vector is treated withcalf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

CKβ-15 protein cDNA capable of expressing active CKβ-15 protein, isisolated. The ends of the portion are modified, if necessary, forcloning into the vector. For instance, 5′overhanging may be treated withDNA polymerase to create blunt ends. 3′ overhanging ends may be removedusing S1 nuclease. Linkers may be ligated to blunt ends with T4 DNAligase.

Equal quantities of the Moloney murine leukemia virus linear backboneand the CKβ-15 protein portion are mixed together and joined using T4DNA ligase. The ligation mixture is used to transform E. coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

Packaging cells are grown in tissue culture to confluent density inDulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the CKβ-15 proteingene is introduced into the packaging cells by standard techniques.Infectious viral particles containing the CKβ-15 protein gene arecollected from the packaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriateincubation period media is harvested from the plates of confluentproducer cells. The media, containing the infectious viral particles, isfiltered through a Millipore filter to remove detached producer cells.The filtered media then is used to infect fibroblast cells. Media isremoved from a sub-confluent plate of fibroblasts and quickly replacedwith the filtered media. Polybrene (Aldrich) may be included in themedia to facilitate transduction. After appropriate incubation, themedia is removed and replaced with fresh media. If the titer of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his, toselect out transduced cells for expansion.

Transformed fibroblasts then may be injected into rats, either alone orafter having been grown to confluence on microcarrier beads, such ascytodex 3 beads. The injected fibroblasts produce CKβ-15 proteinproduct, and the biological actions of the protein are conveyed to thehost.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The disclosures of all patents, patent applications, and publicationsreferred to herein are hereby incorporated by reference.

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) a nucleotide sequence encoding apolypeptide comprising amino acids from about −20 to about 129 in SEQ IDNO:2; (b) a nucleotide sequence encoding a polypeptide comprising aminoacids from about −19 to about 129 in SEQ ID NO:2; (c) a nucleotidesequence encoding a polypeptide comprising amino acids from about 1 toabout 129 in SEQ ID NO:2; (d) a nucleotide sequence encoding apolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC™ Deposit No. 97519; (e) a nucleotide sequence encodingthe mature chemokine β-15 polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC™ Deposit No. 97519; and (f)a nucleotide sequence complementary to any of the nucleotide sequencesin (a), (b), (c), (d), or (e).
 2. The nucleic acid molecule of claim 1wherein said polynucleotide has the complete nucleotide sequence in SEQID NO:1.
 3. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence in SEQ ID NO:1 encoding thechemokine β-15 polypeptide having the complete amino acid sequence inSEQ ID NO:2.
 4. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence in SEQ ID NO:1 encoding themature chemokine β-15 polypeptide having the amino acid sequence in SEQID NO:2.
 5. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the complete nucleotide sequence of the cDNA clonecontained in ATCC™ Deposit No.
 97519. 6. The nucleic acid molecule ofclaim 1 wherein said polynucleotide has the nucleotide sequence encodingthe chemokine β-15 polypeptide having the complete amino acid sequenceencoded by the cDNA clone contained in ATCC™ Deposit No.
 97519. 7. Thenucleic acid molecule of claim 1 wherein said polynucleotide has thenucleotide sequence encoding the mature chemokine β-15 polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC™ Deposit No.
 97519. 8. An isolated nucleic acid molecule comprisinga polynucleotide which hybridizes under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence identical toa nucleotide sequence in (a), (b), (c), (d), (e), or (f) of claim 1wherein said polynucleotide which hybridizes does not hybridize understringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.9. An isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of achemokine β-15 polypeptide having an amino acid sequence in (a), (b),(c), (d), or (e) of claim
 1. 10. A method for making a recombinantvector comprising inserting an isolated nucleic acid molecule of claim 1into a vector.
 11. A recombinant vector produced by the method of claim10.
 12. A method of making a recombinant host cell comprisingintroducing the recombinant vector of claim 11 into a host cell.
 13. Arecombinant host cell produced by the method of claim
 12. 14. Arecombinant method for producing a chemokine β-15 polypeptide,comprising culturing the recombinant host cell of claim 13 underconditions such that said polypeptide is expressed and recovering saidpolypeptide.
 15. An isolated chemokine β-15 polypeptide having an aminoacid sequence at least 95 % identical to a sequence selected from thegroup consisting of: (a) amino acids from about −20 to about 129 in SEQID NO:2; (b) amino acids from about −19 to about 129 in SEQ ID NO:2; (c)amino acids from about 1 to about 129 in SEQ ID NO:2; (d) the amino acidsequence of the chemokine β-15 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC™ Deposit No. 97519;(e) the amino acid sequence of the mature chemokine β-15 polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC™ Deposit No. 97519; and (f) the amino acid sequence of anepitope-bearing portion of any one of the polypeptides of (a), (b), (c),(d), or (e).
 16. An isolated antibody that binds specifically to achemokine β-15 polypeptide of claim
 15. 17. A method for treatment of anindividual in need of an increased level of chemokine β-15 activitycomprising administering to said individual a composition comprising anisolated polypeptide of claim
 15. 18. A method useful during thediagnosis of a disorder of the thymus in an individual comprising: (a)measuring chemokine β-15 gene expression level in cells or body fluid ofsaid individual; (b) comparing the chemokine β-15 gene expression levelof said individual with a standard chemokine β-15 gene expression level,whereby an increase or decrease in the chemokine β-15 gene expressionlevel of said individual compared to said standard expression level isindicative of a thymus disorder.